Means and methods for enhancing the content of sulfur compounds in plants

ABSTRACT

Described are recombinant DNA molecules comprising a nucleic acid molecule encoding a protein having serine acetyltransferase (SAT) activity and optionally a nucleic acid molecule encoding a protein having cysteine-γ-synthase (CγS) activity; wherein said nucleic acid molecule(s) are operably linked to regulatory elements allowing the expression of the nucleic acid molecule(s) in plant cells. Also provided are vectors comprising said recombinant DNA molecules as well as plant cells, plant tissues and plants transformed therewith. In addition, the use of the aforementioned recombinant DNA molecules and vectors in plant cell and tissue culture, plant breeding and/or agriculture is described as well as the use of the aforementioned plants, plant tissue and plant cells for the production of food, feed and additives therefor.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/EP99/04784 which has an Internationalfiling date of Jul. 7, 1999, which designated the United States ofAmerica.

The present invention relates to a recombinant DNA molecule comprising anucleic acid molecule encoding a protein having serine acetyltransferase(SAT) activity and optionally a nucleic acid molecule encoding a proteinhaving cysteine-γ-synthase (CγS) activity; wherein said nucleic acidmolecule(s) are operably linked to regulatory elements allowing theexpression of the nucleic acid molecule(s) in plant cells. The presentinvention also provides vectors comprising said recombinant DNAmolecules as well as plant cells, plant tissues and plants transformedtherewith. The present invention further relates to the use of theaforementioned recombinant DNA molecules and vectors in plant cell andtissue culture, plant breeding and/or agriculture. Furthermore, thepresent invention involves the production of food, feed and additivestherefor comprising the above-described plant cells, plant tissue andplants.

Higher plants use inorganic sulfate in the soil as the major sulfursource for synthesizing the sulfur-containing amino acids cysteine andmethionine. Cysteine biosynthesis in plants has been postulated to playan essential role in the sulfur cycle in nature. Reduced sulfur in theform of cysteine is needed for many different functions in plants(Rennenberg, 1990; Schmidt, 1992). It is essential for the normal plantmetabolism because of connecting serine and methionine metabolism bycarrying the reduced sulfur necessary for methionine biosynthesis(Giovanelli, 1990; Ravanel, 1997; Brunold and Rennenberg, 1997).Additionally, cysteine serves as substrate for other sulfur containingmolecules like certain co-factors, membrane compounds, and as anessential amino acid for protein synthesis (Giovanelli, 1980; Schmidt,1992). Cysteine is also essential as a precursor for the production ofglutathione (GSH) and other stress related metabolites. The demand forcysteine varies during plant development and is also dependend onchanges in the environment, including light, sulfate availability andsome kinds of stress, abiotic or biotic (von Arb and Brunold 1986;Nussbaum, 1988; Delhaize, 1989; Rauser, 1991; Ghisi, 1993; Hell, 1994).

For cysteine biosynthesis, first L-serine has to be activated bytransfer of an acetyl-group from acetyl coenzyme A to form theintermediate O-acetyl-L-serine (OAS). This important reaction iscatalized by serine acetyltransferase (SAT). The activation of serine, akey reaction in the cysteine biosynthetic pathway, has been investigatedat the molecular level only in prokaryotes (Breton, 1990; Monroe, 1990;Evans, 1991; Lai and Baumann, 1992). The synthesis of cysteine in plantsis accomplished by the sulfhydrylation of O-acetyl-L-serine in thepresence of free or bound sulfide, catalized byO-acetylserine(thiol)-lyase (OAS-TL, cysteine synthase, CSase; E C4.2.99.8.) (Schmidt and Jäger, 1990). This reaction has been extensivelyanalysed (Saito, 1992, 1993 and 1994; Rolland, 1993 and 1996;Youssefian, 1993; Noji, 1994; Hell, 1994; Kuske, 1994 and 1996;Takahashi and Saito, 1996). In bacteria SAT and OAS-TL form abifunctional complex called cysteine synthase. In this complex only asmall proportion of the O-acetylserine(thiol)lyase (5%) is associatedwith all the SAT activity (Kredich, 1969). In addition, studies on theregulation of cysteine biosynthesis in bacteria revealed that serineacetyltransferase is sensitive to feedback inhibition by L-cysteine, andthat O-acetylserine (or N-acetylserine) is involved in thetranscriptional activation of several of the cys operon promotors(Ostrowski and Kredich, 1989; Kredich, 1993).

Plant cDNAs encoding serine acetyltransferases have recently been clonedfrom different species (Bogdanova, 1995; Murillo, 1995; Ruffet, 1995;Saito, 1995; Roberts and Wray, 1996). In plants, SAT also exists in acomplex with OAS-TL, suggesting an efficient metabolic channeling fromserine to cysteine by preventing the diffusion of the intermediateO-acetyl-L-serine (Nakamura, 1988; Nakamura and Tamura, 1990; Ruffet,1994; Bogdanova and Hell, 1997; Hesse, 1997). Both SAT and OAS-TL havebeen reported to be localised in plastids, mitochondria and cytosol fromseveral plants, suggesting that the ability to synthesize cysteineappears to be necessary in all cellular compartments with an endogenousprotein biosynthetic capacity (Smith and Thompson, 1969; Smith, 1972;Brunold and Suter, 1982; Lunn, 1990; Rolland, 1992; Ruffet, 1994). InPisum sativum for example, three different isoforms of SAT are existing,and each isoform seems to be specific for a given intracellularcompartment (Ruffet, 1995). Beside these required cellular locations,the fact that cysteine biosynthesis is in complex interaction withuptake and reduction of sulfate, which itself is regulated byphotosynthesis and nitrate assimilation (Anderson, 1990; Brunold, 1993),let suggest an important role of cysteine biosynthesis in sulfurmetabolism in higher plants.

A very important feature of the reaction sequences of cysteine formationis the fact that SAT activity is much lower as compared to the activityof OAS-TL. In seeds and seedlings, OAS-TL is 10 to 20 times more activethan SAT (Smith, 1971; Ngo and Shargool, 1974). In whole leaves theactivity ratio of both enzymes is 100 to 300-fold (Nakamura, 1987),whereas in chloroplasts alone the ratio is up to 345-fold (Ruffet,1994). As has been shown in Allium and spinach, SAT is in comparison toOAS-TL a low abundance enzyme (Nakamura and Tamura, 1990; Ruffet, 1994).Additionally, the availability of OAS was also discussed to be ratelimiting for cysteine synthesis (Neuenschwander, 1991; Ghisi, 1990;Rennenberg 1983; Brunold, 1993; Saito, 1994). SAT activity issignificantly regulated by feedback inhibition of cysteine in watermelon(Saito, 1995). Also on gene expression level SAT regulation takes place.In Arabidopsis thaliana in response to light and sulfur stress SAT mRNAaccumulates by about twofold (Bogdanova, 1995). However, while thefunction and role of SAT, OAS and OAS-TL in the reaction cascade ofcysteine biosynthesis have been subject to a lot of investigationsprevious attempts to alter the rate of cysteine synthesis failed (Saito,1994). Hence, the precise regulation of the cysteine biosyntheticpathway is still not fully understood and part of controversialdiscussion. Therefore, means for the control the sulfur content inplants that may have applications in several aspects of agriculture werehitherto not available.

Thus, the technical problem underlying the present invention was tocomply with the need for means and methods for modulating the content ofsulfur compounds in plants.

The solution to this technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly, the invention relates to a recombinant DNA moleculecomprising

(a) a nucleic acid molecule encoding a protein having serineacetyltransferase (SAT) activity, and optionally

(b) a nucleic acid molecule encoding a protein havingcysteine-γ-synthase (CγS) activity;

wherein said nucleic acid molecule(s) are operably linked to regulatoryelements allowing the expression of the nucleic acid molecule(s) inplant cells.

The term “protein having serine acetyltransferase (SAT) activity”, asused herein, means that said protein is able to transfer an acetyl-groupfrom acetyl coenzyme A to L-serine to form the intermediate of thecysteine biosynthetic pathway O-acetyl-L-serine.

The term “protein having cysteine-γ-synthase (CγS) activity” inaccordance with the present invention denotes a protein capable ofcatalyzing the formation of L-cystathionine or L-homocysteine dependingon the sulfur-containing substrate, L-cysteine or sulfide. This proteinis also known as cystathionine γ-synthase. The terms“cysteine-γ-synthase” and “cystathionine γ-synthase” are usedinterchangeable herein. In plants CγS usually catalyses the firstreaction specific for methionine biosynthesis, namely thegamma-replacement of the phosphoryl substituent of O-phosphohomoserineby cysteine. Thus, cysteine is a major precursor in the biosynthesis ofmethionine in plants.

In accordance with the present invention, the coding sequence of thecysE gene from Escherichia coli (Denk and Böck, 1987), which encodes anenzyme of the cysteine biosynthetic pathway, namely serineacetyltransferase (SAT, EC 2.3.1.30), was introduced into the genome ofpotato plants under the control of the cauliflower mosaic virus (CaMV)35S promoter. To target the protein into the chloroplast cysE wastranslationally fused to the 5′-signal sequence of the small subunit ofrubisco; see Example 1. Successfully transformed plants showed a highaccumulation of the cysE mRNA. Furthermore, crude leaf extracts of theseplants had a significant high SAT-activity, being up to 20 fold higheras compared to wild type plants. The transgenic potato plantsoverexpressing the E. coli gene exhibited elevated levels of cysteineand glutathione (GSH), being two to threefold higher than in controlplants; see Example 2. However, surprisingly the elevation of SAT enzymeactivity and of the substrate for cysteine biosynthesisO-acetyl-L-serine (OAS) had no effect on the expression and on theactivity of O-acetylserine(thiol)-lyase (OAS-TL), the enzyme whichconverts OAS, the product of SAT, to cysteine; see Examples 3 and 4.Both the expression of this gene on RNA level and the enzyme activityremained unchanged compared to wild type plants.

From these experiments, the following conclusions were reached: on onehand the bacterial E. coli SAT expressed in the transgenic potato plantswas accumulated as a catalytically functional protein in thechloroplasts. On the other hand the cellular contents of cysteine andglutathione were significantly increased in leaves of the transgenicplants. The levels of cysteine in one transformant (SAT-48) were nearlythreefold and in another transformant (SAT-26) twofold higher than thoseamounts found in nontransformed control plants, indicating that theexpression of cysE is responsible for the stimulation of cysteinesynthesis. The experiments performed in accordance with the presentinvention also revealed that both transformants had significantlyelevated glutathione levels, being up to twofold higher than in wildtype plants. These unexpected results demonstrate that under normalconditions without any sulfur stress, the endogenous level of SAT is alimiting step in the cysteine biosynthetic pathway, at least in thechloroplast, where the E. coli SAT was targeted to. This means thatunder usual conditions the level of OAS-TL is sufficient for convertingall the OAS produced in the cell and thereby is not limiting for anormal flux of cysteine biosynthesis. The fact, that an overexpressionof the SAT in the transgenic potato plants is able to increase thecysteine and glutathione content further implicates that the steps inthe sulfate assimilation pathway before the incorporation of sulfideinto cysteine, i.e. sulfate uptake, sulfate activation and reduction ofadenosine 5′-phosphosulfate (APS), are under normal conditions also notlimiting for cysteine biosynthesis. The plants seem to possess enoughsulfate uptake capacity and activities to convert sulfate to sulfide forproviding sufficient quantities of reduced sulfur, necessary for theproduction of cysteine. Finally it is worth mentioning, that the resultspresented in accordance with the present invention directly show theconnection between free cysteine and glutathione. The - increased levelsof cysteine in the transgenic potato plants stimulate the biosynthesisof glutathione, leading to levels of the tripeptide, which are up totwofold higher as compared to wild type plants. This suggests, thatglutathione biosynthesis in potato leaves is limited by the availabilityof cysteine. Recently performed experiments with poplar confirm theseresults (Strohm, 1995; Noctor, 1996).

As has been revealed in the experiments performed in accordance with thepresent invention, plants possess enough sulfate uptake capacity andactivities to convert sulfate to sulfid for providing sufficient amountsof reduced sulfur, necessary for the biosynthesis of sulfur containingcompounds. Thus, based on the findings of the present invention, it canbe expected that by introducing cysteine-γ-synthase, the first enzymespecific for the methionine biosynthetic pathway in transgenic plants,the content of methionine could be significantly increased in plantscompared to wild type. In this respect, it is noted that an increase ofthe content of sulfur containing compounds in plants of at least 10%already confer advantageous effects to the plant, for example enhancedtolerance to abiotic stress. Preferably the content of these compoundsis increased by at least about 50%, most preferably 100% andparticularly preferred is the increase of the content of sulfurcontaining compounds of more than 1, preferably 2-fold. Locke, KeystoneMeeting Apr. 6-11, 1997, Abstract 306 (1997) reported the increase ofmethionine by 3 to 5 fold when expressing CγS in plants. Since theintroduction of SAT results in an increase of the substrate of CγS it isexpected by introducing the CγS in SAT expressing plants of theinvention or vice versa the increase of the content of sulfur containingcompounds is further increased about 1 to 10 fold, preferably 5 to 10fold or higher.

In a preferred embodiment of the recombinant DNA molecule of theinventiori, said protein having SAT activity is a serineacetyltransferase derived from prokaryotes or archaebacteria.Prokaryotic organisms may include gram negative as well as gram positivebacteria such as, for example, E. coli, S. typhimurium, Serratiamarcescens, Bacillus subtilis and various species within the generaPseudomonas, Streptomyces and Staphylococcus, although others may alsobe employed as well. For example, nucleic acid molecules encodingproteins having SAT activity can be obtained from the prior art (e.g.,Bogdanova, FEBS Lett. 358 (1995), 43-47; Denk, J. Gen. Microbiol. 133(1987), 515-525; Evans, J. Bacteriol. 173 (1991), 5457-5469).

In general the nucleic acid molecule encoding a protein having CγSactivity can be derived from any material source, for example, from anyplant possessing such molecules, preferably form monocotyledonous ordicotyledonous plants, in particular from any plant of interest inagriculture, horticulture or wood culture, such as crop plants, namelythose of the family Poaceae, any starch producing plants, such aspotato, maniok, leguminous plants, oil producing plants, such as oilseedrape, linenseed, etc., plants using polypeptide as storage substances,such as soybean, plants using sucrose as storage substance, such assugar beet or sugar cane, trees, ornamental plants etc. or plantsbelonging to the family Gramineae. Nucleic acid molecules encodingcysteine γ-synthase are described in the prior art, for example, inRavanel, Biochem. J. 331 (1998), 639-648 and references cited therein.

Furthermore, nucleic acid molecules can be used hybridizing to theabove-described nucleic acid molecules and encoding a protein having SATand CγS activity, respectively. Such nucleic acid molecules can beisolated, e.g., from libraries, such as cDNA or genomic libraries bytechniques well known in the art. For example, hybridizing nucleic acidmolecules can be identified and isolated by using the above-describednucleic acid molecules known in the art or fragments thereof orcomplements thereof as probes to screen libraries by hybridizing withsaid molecules according to standard techniques. Possible is also theisolation of such nucleic acid molecules by applying the polymerasechain reaction (PCR) using as primers oligonucleotides derived form theabove-described nucleic acid molecules. Nucleic acid molecules whichhybridize with any of the aforementioned nucleic acid molecules alsoinclude fragments, derivatives and allelic variants of theabove-described nucleic acid molecules that encode a protein having SATor CγS activity or biologically active fragments thereof. Fragments areunderstood to be parts of nucleic acid molecules long enough to encodethe described protein or a fragment thereof having the biologicalactivity as defined above.

The term “derivative” means in this context that the nucleotide sequenceof these nucleic acid molecules differs from the sequences of theabove-described nucleic acid molecules in one or more nucleotidepositions and are highly homologous to said nucleic acid molecules.Homology is understood to refer to a sequence identity of at least 40%,particularly an identity of at least 60%, preferably more than 80% andstill more preferably more than 90%. The deviations from the sequencesof the nucleic acid molecules described above can, for example, be theresult of nucleotide substitution(s), deletion(s), addition(s),insertion(s) and/or recombination(s) either alone or in combination,that may naturally occur or be produced via recombinant DNA techniqueswell known in the art; see for example, the techniques described inSambrook, Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory (1989) N.Y. and Ausubel, Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Interscience, N.Y.(1989). Homology further means that the respective nucleic acidmolecules or encoded proteins are functionally and/or structurallyequivalent. The nucleic acid molecules that are homologous to thenucleic acid molecules described above and that are derivatives of saidnucleic acid molecules are, for example, variations of said nucleic acidmolecules which represent modifications having the same biologicalfunction, in particular encoding proteins with the same or substantiallythe same biological activity as defined herein. They may be naturallyoccurring variations, such as SAT and CγS protein encoding sequencesfrom other prokaryotes and plants, respectively, or mutations. Thesemutations may occur naturally or may be obtained by mutagenesistechniques, see supra. The allelic variations may be naturally occurringallelic variants as well as synthetically produced or geneticallyengineered variants; see supra. For example, the amino acid sequences ofplant SATs share significant similarities with bacterial serineacetyltransferases (Vuorio, 1994; Bogdanova and Hell, 1997). The mostconserved region within all SATs, both from plants and bacteria, islocated at the C-terminus and has been suggested to confer thetransferase activity (Vaara, 1992; Vuorio, 1994). In this conservedregion a hexapeptide motif is present that has been proposed as acatalytic domain in bacterial acetyltransferases and that recently hasbeen demonstrated to be also present in the OAS-TL/SAT contact region inthe cysteine synthase complex from Arabidopsis thaliana (Bogdanova andHell, 1997). In addition, nucleic acid molecules can be employed inaccordance with the present invention that encode homologs or analogs ofthe above described proteins having SAT or CγS activity but whereotherwise unrelated to those proteins. For example, malY of E. coliencodes an enzyme that is involved in the uptake and metabolism ofmaltose and maltodextrins of the E. coli maltose system but has inaddition the enzyme activity of cystathionine β-lyase; see Zdych, J.Bacteriol. 177 (1995), 5035-5039. However, said proteins are nothomologous to each other based on amino acid sequence homology analysis.Such proteins that do not display significant homologies to common SATor CγS proteins can be identified by a person skilled in the art usingtechniques well known in the art, for example, via complementation ofmutant genes involved in the cysteine or methionine biosyntheticpathway, for example, in corresponding mutant E. coli strains; see alsoZdych, supra.

The proteins encoded by the various derivatives, variants, homologs oranalogs of the above-described nucleic acid molecules may share specificcommon characteristics, such as molecular weight, immunologicalreactivity, conformation, etc., as well as physical properties, such aselectrophoretic mobility, chromatographic behavior, sedimentationcoefficients, pH optimum, temperature optimum, stability, solubility,spectroscopic properties, etc. All these nucleic acid molecules andderivatives can be employed in accordance with the present invention aslong as the enzymatic activity of the encoded protein remainssubstantially unaffected in kind, namely that the protein has SAT andCγS activity, respectively, as defined above.

In a preferred embodiment of the recombinant DNA molecule of theinvention, the protein having CγS-activity is cysteine-γ-synthase frompotato, tabacco, tomato, rape seed or Arabidopsis; see, e.g., Kim andLeustek, Plant Mol. Biol. 32 (1996), 1117-1124.

In a preferred embodiment of the recombinant DNA molecule of theinvention, the nucleic acid molecule of (a) and/or (b) is operablylinked to a nucleotide sequence encoding a transit peptide capable ofdirecting the protein(s) into a desired cellular compartment. Thenucleic acid molecule present in the recombinant DNA molecule accordingto the invention can be modified in such a way that the encoded proteinis located in any desired compartment of the plant cell. These includethe endoplasmatic reticulum (KDEL, Schouten, Plant Mol. Biol. 30 (1996),781-793), the vacuole (Neuhaus, PNAS 88 (1991), 10362-10366), themitochondria (Chaumont, Plant Mol. Biol. 24 (1994), 631-641), theplastids (Fuhr, EMBO J. 5 (1986), 2063-2071), the apoplast (vonSchaewen, EMBO J. 9 (1990), 3033-3044), the cytoplasm etc. Methods howto carry out these modifications and signal sequences ensuringlocalization in a desired compartment are well known to the personskilled in the art. Preferably, said cellular compartment is a plastid.As is described in the appended examples, the protein having SATactivity was targeted into the chloroplast via the translational fusionto the 5′-signal sequence of the small subunit of rubisco.Advantageously, the protein having CγS activity may be coexpressed inthe same cellular compartment, for example in the chloroplast andshould, therefore, provide for significant increase of methioninecontent in plant leafs as well.

The recombinant DNA molecule of the invention comprises regulatorysequences allowing for the expression the nucleic acid molecules inplant cells. Preferably, said regulatory elements comprise a promoteractive in plant cells. Expression comprises transcription of the nucleicacid molecule into a translatable mRNA. Regulatory elements ensuringexpression in plant cells are well known to those skilled in the art.

These regulatory elements may be heterologous or homologous with respectto the nucleic acid molecule to be expressed as well with respect to theplant species to be transformed. In general, such regulatory elementscomprise a promoter active in plant cells. To obtain expression in alltissues of a transgenic plant, preferably constitutive promoters areused, such as the 35S promoter of CaMV (Odell, Nature 313 (1985),810-812) or promoters of the polyubiquitin genes of maize (Christensen,Plant Mol. Biol. 18 (1982), 675-689). In order to achieve expression inspecific tissues of a transgenic plant it is possible to use tissuespecific promoters (see, e.g., Stockhaus, EMBO J. 8 (1989), 2245-2251).Known are also promoters which are specifically active in tubers ofpotatoes or in seeds of different plant species, such as maize, Vicia,wheat, barley etc. Inducible promoters may be used in order to be ableto exactly control expression. An example for inducible promoters arethe promoters of genes encoding heat shock proteins. Alsomicrospore-specific regulatory elements and their uses have beendescribed (WO96/16182). Furthermore, the chemically inducibleTest-system may be employed (Gatz, Mol. Gen. Genet. 227 (1991);229-237). Further suitable promoters are known to the person skilled inthe art and are described, e.g., in Ward (Plant Mol. Biol. 22 (1993),361-366). The regulatory elements may further comprise transcriptionaland/or translational enhancers functional in plants cells. A planttranslational enhancer often used is, e.g., the CaMV omega sequencesand/or the inclusion of an intron (Intron-1 from the Shrunken gene ofmaize, for example) that has been shown to increase expression levels byup to 100-fold. (Maiti, Transgenic Research 6 (1997), 143-156; Ni, PlantJournal 7 (1995), 661-676). Furthermore, the regulatory elements mayinclude transcription termination signals, such as a poly-A signal,which lead to the addition of a poly A tail to the transcript which mayimprove its stability. The termination signals usually employed are fromthe Nopaline Synthase gene or from the CaMV 35S RNA gene.

In a preferred embodiment of the recombinant DNA molecule of theinvention, said promoter is inducible or constitutively expressed and/oris a cell, tissue or organ specific promoter. Preferably, said promoteris tuber-specific, seed-specific, endosperm-specific, embryo-specific,or phloem-specific. Examples for such promoters include but are notlimited to patatin promoter B33 (tuber-specific, Rocha-Sosa, EMBO J. 8(1989), 23), phaseolin promoter (seed-specific, Karchi, Plant J. 3(1993), 721-727), HMW glutenin promoter (endosperm-specific, Helford,Theor. Appl. Genet. 75 (1987), 117-126), α, β-conglycin promoter(embryo-specific, Fujiwara, Plant Cell Reports 9 (1991), 602-606), rolCpromoter (phloem-specific, Lerchl, Plant Cell 7 (1995), 259-270).

The present invention also relates to vectors, particularly plasmids,cosmids, viruses, bacteriophages and other vectors used conventionallyin genetic engineering that contain at least one recombinant DNAmolecule according to the invention. Methods which are well known tothose skilled in the art can be used to construct various plasmids andvectors; see, for example, the techniques described in Sambrook,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory(1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y. (1989).Alternatively, the recombinant DNA molecules and vectors of theinvention can be reconstituted into liposomes for delivery to targetcells.

Advantageously the above-described vectors of the invention comprises aselectable and/or scorable marker. Selectable marker genes useful forthe selection of transformed plant cells, callus, plant tissue andplants are well known to those skilled in the art and comprise, forexample, antimetabolite resistance as the basis of selection for dhfr,which confers resistance to methotrexate (Reiss, Plant Physiol. (LifeSci. Adv.) 13 (1994), 143-149); npt, which confers resistance to theaminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella,EMBO J. 2 (1983), 987-995) and hygro, which confers resistance tohygromycin (Marsh, Gene 32 (1984), 481-485). Additional selectable geneshave been described, namely trpB, which allows cells to utilize indolein place of tryptophan; hisD, which allows cells to utilize histinol inplace of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988),8047); mannose-6-phosphate isomerase which allows cells to utilizemannose (WO 94/20627) and ODC (ornithine decarboxylase) which confersresistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.)or deaminase from Aspergillus terreus which confers resistance toBlasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995),2336-2338). Useful scorable marker are also known to those skilled inthe art and are commercially available. Advantageously, said marker is agene encoding luciferase (Giacomin, Pl. Sci. 116 (1996), 59-72;Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes,FEBS Lett. 389 (1996), 44-47) or β-glucuronidase (Jefferson, EMBO J. 6(1987), 3901-3907). This embodiment is particularly useful for simpleand rapid screening of cells, tissues and organisms containing a vectorof the invention.

The recombinant DNA molecules according to the invention are inparticular useful for the genetic manipulation of plant cells, planttissue and plants in order to enhance their content of sulfur containingcompounds and to obtain plants with modified, preferably with improvedor useful phenotypes. Thus, the present invention provides for a methodfor the production of transgenic plants, plant cells or plant tissuecomprising the introduction of at least one recombinant DNA molecule orvector of the invention into the genome of said plants, plant cells orplant tissue.

Methods for the introduction of foreign DNA into plants are also wellknown in the art. These include, for example, the transformation ofplant cells, plant tissue or plants with T-DNA using Agrobacteriumtumefaciens or Agrobacterium rhizogenes, the fusion of protoplasts,direct gene transfer (see, e.g., EP-A 164 575), injection,electroporation, biolistic methods like particle bombardment and othermethods known in the art. The vectors used in the method of theinvention may contain further functional elements, for example “leftborder”- and “right border”-sequences of the T-DNA of Agrobacteriumwhich allow for stably integration into the plant genome. Furthermore,methods and vectors are known to the person skilled in the art whichpermit the generation of marker free transgenic plants, i.e. theselectable or scorable marker gene is lost at a certain stage of plantdevelopment or plant breeding. This can be achieved by, for examplecotransformation (Lyznik, Plant Mol. Biol. 13 (1989), 151-161; Peng,Plant Mol. Biol. 27 (1995), 91-104) and/or by using systems whichutilize enzymes capable of promoting homologous recombination in plants(see, e.g., WO97/08331; Bayley, Plant Mol. Biol. 18 (1992), 353-361);Lloyd, Mol. Gen. Genet. 242 (1994), 653-657; Maeser, Mol. Gen. Genet.230 (1991), 170-176; Onouchi, Nucl. Acids Res. 19 (1991), 6373-6378).Methods for the preparation of appropriate vectors are described by,e.g., Sambrook (Molecular Cloding; A Laboratory Manual, 2nd Edition(1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Suitable strains of Agrobacterium tumefaciens and vectors as well astransformation of Agrobacteria and appropriate growth and selectionmedia are well known to those skilled in the art and are described inthe prior art (GV3101 (pMK90RK), Koncz, Mol. Gen. Genet. 204 (1986),383-396; C58C1 (pGV 3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777;Bevan, Nucleic. Acid Res. 12(1984), 8711; Koncz, Proc. Natl. Acad. Sci.USA 86 (1989), 8467-8471; Koncz, Plant Mol. Biol. 20 (1992), 963-976;Koncz, Specialized vectors for gene tagging and expression studies. In:Plant Molecular Biology Manual Vol 2, Gelvin and Schilperoort (Eds.),Dordrecht, The Netherlands: Kluwer Academic Publ. (1994), 1-22; EP-A-120516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4,1-46; An, EMBO J. 4 (1985), 277-287). Although the use of Agrobacteriumtumefaciens is preferred in the method of the invention, otherAgrobacterium strains, such as Agrobacterium rhizogenes, may be used,for example if a phenotype conferred by said strain is desired.

Methods for the transformation using biolistic methods are well known tothe person skilled in the art; see, e.g., Wan, Plant Physiol. 104(1994), 37-48; Vasil, Bio/Technology 11 (1993), 1553-1558 and Christou(1996) Trends in Plant Science 1, 423-431. Microinjection can beperformed as described in Potrykus and Spangenberg (eds.), Gene TransferTo Plants. Springer Verlag, Berlin, N.Y. (1995). The transformation ofmost dicotyledonous plants is possible with the methods described above.But also for the transformation of monocotyledonous plants severalsuccessful transformation techniques have been developed. These includethe transformation using biolistic methods as, e.g., described above aswell as protoplast transformation, electroporation of partiallypermeabilized cells, introduction of DNA using glass fibers, etc.

In general, the plants, plant cells and plant tissue which can bemodified with a recombinant DNA molecule or vector according to theinvention and which show (over)expression of a proteins having SAT andoptionally CγS activity, respectively, can be derived from any desiredplant species. They can be monocotyledonous plants or dicotyledonousplants, preferably they belong to plant species of interest inagriculture, wood culture or horticulture interest, such as crop plants(e.g. maize, rice, barley, wheat, rye, oats etc.), potatoes, oilproducing plants (e.g. oilseed rape, sunflower, peanut, soybean, etc.),cotton, sugar beet, sugar cane, leguminous plants (e.g. beans, peasetc.), wood producing plants, preferably trees, etc.

Thus, the present invention relates also to transgenic plant cells whichcontain stably integrated into the genome a nucleic acid moleculeaccording to the invention linked to regulatory elements which allow forexpression of the nucleic acid molecule in plant cells and wherein thenucleic acid molecule is foreign to the transgenic plant cell. By“foreign” it is meant that the nucleic acid molecule is eitherheterologous with respect to the plant cell, this means derived from acell or organism with a different genomic background, or is homologouswith respect to the plant cell but located in a different genomicenvironment than the naturally occurring counterpart of said nucleicacid molecule. This means that, if the nucleic acid molecule ishomologous with respect to the plant cell, it is not located in itsnatural location in the genome of said plant cell, in particular it issurrounded by different genes. In this case the nucleic acid moleculemay be either under the control of its own promoter or under the controlof a heterologous promoter. The vector or recombinant DNA moleculeaccording to the invention which is present in the host cell may eitherbe integrated into the genome of the host cell or it may be maintainedin some form extrachromosomally.

Alternatively, a plant cell having (a) nucleic acid molecule(s) encodinga protein having SAT and optionally CγS activity present in its genomecan be used and modified such that said plant cell expresses theendogenous gene(s) corresponding to these nucleic acid molecules underthe control of an heterologous promoter and/or enhancer elements. Theintroduction of the heterologous promoter and mentioned elements whichdo not naturally control the expression of a nucleic acid moleculeencoding either of the above described proteins using, e.g., genetargeting vectors can be done according to standard methods, see supraand, e.g., Hayashi, Science 258 (1992), 1350-1353; Fritze and Walden,Gene activation by T-DNA tagging. In Methods in Molecular biology 44(Gartland, K. M. A. and Davey, M. R., eds). Totowa: Human Press (1995),281-294) or transposon tagging (Chandlee, Physiologia Plantarum 78(1990), 105-115). Suitable promoters and other regulatory elements suchas enhancers include those mentioned hereinbefore.

The presence and expression of the nucleic acid molecule(s) present inthe recombinant DNA molecule or vector in the transgenic plant cellslead(s) to the synthesis of proteins which has (have) an influence on,e.g., stress resistance of the plant cells and leads to correspondingphysiological and phenotypic changes in plants containing such cells. Asis described in the appended examples, plants constitutively expressingE. coli SAT display high levels of cysteine. Additionally, the contentof the tripeptide glutathione (γ-glutamylcysteineylglycine) wasconsiderably higher than in wild type plants, because this compound isthe main storage form of reduced sulfur in plant kingdom and serves asthe major sink of produced cysteine. Glutathione plays not only anessential role in the regulation of sulfur nutrition, but is also animportant factor in the defense of plants against various forms ofstress, including high light intensities, drought, cold, heat andmineral deficiency (Smith, 1990; Rennenberg and Brunold, 1994). Thetripeptide is synthesized in plants as well as in other organisms in twoenzyme-catalyzed reactions from the constituent amino acids (Meister andAnderson, 1983; Rennenberg, 1995).

As discussed above, cysteine-γ-synthase is capable of using cysteine asthe sulfur-containing substrate. Therefore, since it has beendemonstrated in the course of the present invention, that transgenicplants overexpressing a protein having SAT activity have a considerablyhigher content of cysteine, coexpression of a nucleic acid moleculeencoding a protein having CγS activity should provide for a synergisticeffect in the production of methionine, since both the key-enzyme of themethionine biosynthetic pathway and its substrate are overproduced inthe plants. Therefore, in a preferred embodiment of the invention, saidplant cell comprises (a) recombinant DNA molecule(s) comprising

(a) a nucleic acid molecule encoding a protein having serine acetyltransferase (SAT) activity, and

(b) a nucleic acid molecule encoding a protein havingcysteine-γ-synthase (CγS) activity;

wherein said nucleic acid molecule(s) are operably linked to regulatoryelements as described above.

As is immediately evident to the person skilled in the art, therecombinant DNA molecule of the present invention can carry the nucleicacid molecules as defined in (a) and (b) either alone or in combination.The same applies to the above described vectors of the present inventionas Well as to plant cells, plant tissue and plants transformedtherewith. Likewise, said nucleic acid molecules may be under thecontrol of the same regulatory elements or may be separately controlledfor expression. In this respect, the person skilled in the art willreadily appreciate that the nucleic acid molecules encoding a proteinhaving SAT and CγS activity, respectively, can be expressed in the formof a single mRNA as transcriptional and optionally translationalfusions. This means that the proteins having SAT and CγS activity,respectively, are produced as separate polypeptides or in the latteroption as a fusion polypeptide that is further processed into theindividual proteins, for example via a cleavage site for proteinasesthat has been incorporated between the amino acid sequences of bothproteins. Of course, the proteins having SAT and CγS activity,respectively, may also be expressed as a bi- or multifunctionalpolypeptide, preferably disposed by a peptide linker whichadvantageously allows for sufficient flexibility of both proteins.Preferably said peptide linker comprises plural, hydrophilic,peptide-bonded amino acids of a length sufficient to span the distancebetween the C-terminal end of one of said proteins and the N-terminalend of the other of said proteins when said polypeptide assumes aconformation suitable for biological activity of both proteins whendisposed in aqueous solution in the plant cell. Furthermore, therecombinant DNA molecules and vectors of the invention may comprisefurther genes encoding other proteins involved in cysteine and/ormethionine biosynthesis. Examples for the above-described expressionstrategies can be found in the literature, e.g., for dicistronic mRNA(Reinitiation) in Hefferon, J. Gen. tirol. 78 (1997), 3051-3059, fusionproteins are described in Brinck-Peterson, Plant Mol. Biol. 32 (1996),611-620 and Hotze, FEBS Lett. 374 (1995), 345-350; bifunctional proteinsare discussed in Lamp, Biochem. Biophys. Res. Com. 244 (1998), 110-114and Dumas, FEBS Lett. 408 (1997), 156-160 and for linker peptide andprotease it is referred to Doskeland, Biochem. J. 313 (1996), 409-414.

In a preferred embodiment of the invention, the transgenic plant cellcomprises a selectable marker. As described above, various selectablemarkers can be employed in accordance with the present invention.Advantageously, selectable markers may be used that are suitable fordirect selection of transformed plants, for example, thephophinothricin-N-acetyltransferase gene the gene product of whichdetoxifies the herbicide L-phosphinothricin (glufosinate or BASTA); see,e.g., De Block, EMBO J. 6 (1987), 2513-2518 and Drnge, Planta 187(1992), 142-151.

Furthermore, the present invention also relates to transgenic plants andplant tissue comprising the above-described transgenic plant cells orobtainable by the above described method. These may show, for example,improved stress resistance. Preferably, the level of glutathione,cysteine and/or methionine in the transgenic plant of the invention isincreased compared to a wild type plant. An increase of the level ofglutathione, cysteine and/or methionine is understood to refer to anelevated content of any one of the above cited sulfur containingcompounds either alone or in combination in the transgenic plant cells,plant tissue or plants of the present invention in the order of at leastabout 10% compared to the corresponding non-transformed wild type plantcell, plant tissue or plant, which already provides for beneficialeffects on the vitality of the plant such as, e.g., improved stresstolerance. Advantageously, the content of the above-described compoundsis increased by at least about 50%, preferably by more than about 75%,particularly preferred at least about or more than 100% and still morepreferably more than about 200%. Considering the content of cysteine,methionine and glutathione in combination even an 10- to 20-foldincrease of sulfur containing compounds compared to the level of freecysteine in wild type plants can be achieved although higher increasesof sulfur containing compounds in the plants of the present inventionare envisaged as well.

Advantageously, the level of free cysteine in the plants of the presentinvention is about higher than 20 nmol per gram fresh weight (gfw) ofleaf tissue, preferably higher than 30 nmol/gfw and more preferablyhigher than 40 nmol/gfw, see also Example 2. Furthermore, the content ofglutathione in the plants of the present invention can be preferablyhigher than 350 nmol/gfw, more preferably higher than 500 nmol/gfw. Thecontent of methionine can be increased by at least about 5 fold,preferably more than 10 fold.

In yet another aspect, the invention also relates to harvestable partsand to propagation material of the transgenic plants according to theinvention which contain transgenic plant cells described above, i.e. atleast one recombinant DNA molecule or vector according to the inventionand/or which are derived from the above described plants and displayincreased levels of sulfur containing compounds as described supra.Harvestable parts can be in principle any useful parts of a plant, forexample, leaves, stems, fruit, seeds, roots etc. Propagation materialincludes, for example, seeds, fruits, cuttings, seedlings, tubers,rootstocks etc.

Furthermore, the present invention relates to use of at least onerecombinant DNA molecule or vector of the invention for the productionof transgenic plants which display an increased level of glutathione,cysteine and/or methionine. Preferably, said increased level ofmethionine or cysteine results in accelerated maturation processes,altered flowers and/or pathogen resistance.

The constitutive expression of the E. coli SAT cysE gene in transgenicpotato plants directly demonstrate in vivo, that the SAT-catalizedreaction is indeed rate-limiting in the plant cysteine biosyntheticpathway, as is shown by the high levels of cysteine in thetransformants; see the appended examples. Furthermore, as discussedabove, it is expected that the corresponding high levels of glutathionein the transgenic plants are able to confer resistance against variousforms of stress. In plants glutathione plays an important role in thedefense against active oxygen species, xenobiotics, heavy metals andother forms of stresses including drought, heat and mineral deficiency(Alscher, 1989; Smith, 1990; Schmidt and Jäger, 1992; Rennenberg andBrunold, 1994; Rennenberg, 1995). Knowledge about this is also ofpractical importance. Higher resistance of plants against active oxygenspecies may play a very important role in future, thinking of theelevated ozon concentrations in the atmosphere. Furthermore, anincreased tolerance against xenobiotics, for example herbicides, as aresult of higher glutathione levels in the plants of the invention isreasonable. Moreover, a possible strategy is to construct transgenicplants which are able to grow on higher concentrations of heavy metalions and which therefore could be used for bioremidation. Furthermore,the recombinant DNA molecules and vectors according to the invention maybe useful for the alteration or modification of plant/pathogeninteraction. The term “pathogen” includes, for example, bacteria,viruses and fungi as well as protozoa.

As discussed above, the transgenic plant cells, tissue and plants of theinvention can be used to ameliorate the toxic effects of pollutants insoil including the water economy. Pollutants may be naturally present orbe caused by mining, manufacturing and urban activities. Such pollutantscomprise compounds which may inactivate sulfur containing proteins, inparticular enzymes or act as antagonists or inhibitors in the cysteineand/or methionine biosynthetic pathway. Examples for such antagonists orinhibitors are herbicides, fungicides, pesticides or particularly metalions, e.g., Hg²⁺. For example, due to the elevated levels of GSHconferred by the expression of the nucleic acid molecules contained inthe recombinant DNA molecules and vectors of the present invention inthe above described plants it is possible to employ soil for agriculturewhich is otherwise not suitable because of the presence of, for example,toxic compounds which interfere with the sulfur containing enzymes andthus with plant growth. This is in particular true for soil whichcontains large amounts of heavy metals. Moreover, the plant cells, planttissue and plants of the present invention can be used for remediationof soil contaminated with pollutants. An advantageous side effect isthat by, for example, increased metal tolerance due to the presence ofthe recombinant DNA molecule or vector of the invention, the plantcells, plant tissue and plant of the present invention can be used for“biomining” (Cunningham, TIBTECH 13 (1995), 393-397). This means thatthe plants, plant tissue and plant cells of the present invention can beused for phytoextraction of metals such as mercury, nickel and copper.Furthermore, as mentioned before a higher content of GSH in the plantcells, plant tissue and plants of the invention can provide forincreased resistance. In addition, the high level of GSH present in theabove-described plant cells or plant tissues and plants is expected toresult in improved growth of seedlings and biomass production. Insummary, increase of the GSH content has manyfold effects on thevitality of plants and is of particular interest for the plant breeder.

Furthermore, overexpression of cysteine-γ-synthase in plants incombination with the SAT encoding nucleic acid molecule in transgenicplant cells, plant tissue and plants of the invention results in anincrease of free as well as bound methionine in the plants, which isparticularly advantageous for food and feed, since methionine is anessential amino acid and usually present only in low amounts in food andfeed stuffs that, therefore, are not sufficient for the supply of thisamino acid to humans and animals. Thus, the plant cells, plant tissuesand plants as well as the harvestable parts and/or propagation materialthereof can be used as feed or food or as additives therefor.Furthermore, the increase of methionine can enhance maturationprocesses, flowering as well as pathogen resistance. Also, highmethionine contents in plants or harvestable parts thereof as well aspropagation material of the invention significantly contribute to theattractive taste of various food products such as baked bread and roastcoffee and the like.

Thus, the present invention relates in a further embodiment to use of anucleic acid molecule encoding a protein having SAT activity or at leastone recombinant DNA molecule or vector of the invention, a plant cell, aplant or plant tissue of the invention or, harvestable parts orpropagation material thereof for the production of food, animal feed,for the improvement of pathogen resistance, for conferring heavy metallor herbicide tolerance, for improving biomass production, for enhancinggrowth of seedlings, for conferring tolerance against biotic or abioticstress, or for improving the flavour and/or taste of food or feed. Theuse of the recombinant DNA molecule or vector of the invention forconferring herbicide tolerance includes, for example, their use asselectable markers in plants according to other systems which employ(over)expression of enzymes capable of conferring tolerance (i.e.resistance) to plant cell killing effects of herbicides. An example forsuch a system is the overexpression of the enzyme5-enolpyruvylshikimate-3-phosphate (EPSP) synthase that conferstolerance to the herbicide glyphosphate. In a similar way, therecombinant DNA molecules and vectors of the invention can be used forconferring tolerance against compounds that act on sulfur containingenzymes via, e.g., sequestering the compound that is responsible forinhibition of said enzymes by the high content of cysteine, GSH and/ormethionine or by peptides and proteins that are present in higher levelsdue to the elevated content of the sulfur containing amino acids.

As described above, the nutrional value of the plants, plant tissue andplant cells of the invention as well as harvestable parts andpropagation material of such plants is considerably improved due to theincreased content of sulfur containing compounds. Therefore, the presentinvention also relates to feed and food or additives therefor comprisingplant cells, plant tissue, plant, harvestable parts or propagationmaterial of the invention. These feed, food and additives preferablyhave increased contents of cysteine, methionine and/or glutathione suchas described above.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the methods, uses and compounds to be employed inaccordance with the present invention may be retrieved from publiclibraries, using for example electronic devices. For example the publicdatabase “Medline” may be utilized which is available on the Internet,for example under http://www.ncbi.nlm.nih.gov/PubMed/medline.html.Further databases and addresses, such as http://www.ncbi.nlm.nih.gov/,http://www.infobiogen.fr/,http://www.fmi.ch/biology/research_tools.html, http://www.tigr.org/, areknown to the person skilled in the art and can also be obtained using,e.g., http://www.lycos.com. An overview of patent information inbiotechnology and a survey of relevant sources of patent informationuseful for retrospective searching and for current awareness is given inBerks, TIBTECH 12 (1994), 352-364.

THE FIGURES SHOW

FIG. 1: (a) Northern blot analysis of total RNA extracted from leaves offive independent trangenic (SAT-65, 26, 48, 3 and 71) and two wild typeplants (Control a and b). The blot was probed with ³²P-labeled cysE DNAfrom E. coli. The lanes contained 15 μg of total RNA each.

(b) Maximum catalytic activity of SAT in leaves of five independenttransgenic (SAT-65, 26, 48, 3 and 71) and two wild type plants (Controla and b). The specific activity of crude extracts is given in pmolproduced CoA per min and μg total protein. Error bars represent standarddeviation (<10%). N=4 independent measurements.

FIG. 2: (a) Endogenous levels of cysteine in leaves of 6-week oldtransgenic (SAT-48 and SAT-26) and wild type plants (Control a and b).Amounts of cysteine are given in nmol per gfw. Error bars representstandard deviation (<20). N=18; 6 independent plants per transgenicline, 3 independent measurements per plant.

(b) Endogenous levels of glutathione in leaves of 6-week old transgenic(SAT-48 and SAT-26) and wild type plants (Control a and b). Amounts ofglutathione are given in nmol per gfw. Error bars represent standarddeviation (<10). N=18; 6 independent plants per transgenic line, 3independent measurements per plant.

FIG. 3: Northern blot analysis of total RNA extracted from leaves offive independent transgenic (SAT-65, 26, 48, 3 and 71) and two wild typeplants (Control a and b). The blot was probed with ³²P-labeled plastidicpotato OAS-TL cDNA (p) and also with a cytosolic isoform (c). The lanescontained 15 μg of total RNA each.

FIG. 4: In vitro OAS-TL activity in leaves of transgenic (SAT-48 and 26)and wild type plants (Control a and b). The specific activity of crudeextracts is given in pmol produced cysteine per min and μg totalprotein. Error bars represent standard deviation (<10%). N=4 independentmeasurements.

The Examples illustrate the invention:

EXAMPLE 1 Screening for Transgenic Potato Plants Containing the E. coliSAT mRNA

To target SAT from E. coli to chloroplasts of plants a gene fusion witha Rubisco transit peptide from Arabidopsis was constructed. SAT from E.coli genomic DNA was amplified by PCR using two syntheticoligonucleotides (EcSAT-N: 5′-GAG AGA CCA TGG CGT GTG AAG AAC TGG AAA(SEQ ID NO: 1), EcSAT-C: 5′-GAG AGA TCT AGA TTA GAT CCC ATC CCC ATA (SEQID NO: 2)) Double stranded DNA was digested with Nco I and Xba I andcloned behind the transit peptide. The fused gene product was insertedas Asp 718/Xho I fragment into a with Asp 718/Sal I predigested binaryvector (Höfgen and Willmitzer, 1990) under the contol of the 35S-CaMVpromoter. The plasmid was introduced into potato via Agrobacteriumtumefaciens (Solanum tuberosum cv Dësirëe) as described by Rocha-Sosa etal. (1989). Solanum tuberosum cv Désirée was obtained from VereinigteSaatzuchten eG (Ebstorf, Germany). Wild type and transgenic plants werekept in tissue culture under a 16-hours-light/8-hours-dark period onMurashige and Skoog medium (Murashige and Skoog, 1962) supplemented with2% (w/v) sucrose at 22° C. In the greenhouse, plants were grown at 22°C. during the light period (16 hours) and 15° C. during the dark period(8 hours). The plants were cultivated in separate pots and wateredcontinuously.

Transgenic potato plants maintained in tissue culture were visuallyindistinguishable from nontransformed control plants. To screen forplants expressing the E. coli SAT mRNA, fifty plants were randomlyselected for taking leaf samples. These samples were subjected to RNAgel blot analysis using a radioactively labelled E. coli SAT cDNA as aprobe. For RNA isolation, plant leaf material was frozen in liquidnitrogen directly after harvest. Total RNA was extracted from the frozenmaterial according to Logemann et al. (1987). After denaturation at 65°C. the total RNA was separated under denaturing conditions by gelelectrophoresis (Lehrach, 1977) and then transferred to nylon membranes.Northern hybridisation was performed at an appropriate temperature asdescribed by Amasino (1986). The northern blots were washed three timesfor 30 min at 55° C. in 0,5×SSC; 0,2% SDS. ³²P-labelling of thefragments was performed with the “Multiprime DNA-labelling-Kit”(Amersham Buchler, Braunschweig, Germany). Five independenttransformants accumulating high amounts of the foreign SAT mRNA, wereselected for further analysis and transferred into the greenhouse.Repeated Northern analysis revealed, that also under greenhouseconditions the transformants accumulated high amounts of the foreignmRNA (FIG. 1a). The length of the transcript detected in the transgenicpotato plants (˜1050 basepairs) was in agreement with the lengthreported for the cysE gene, namely 819 bp (Denk and Böck, 1987) and theused signal sequence of rubisco (˜240 bp). For measuring the enzymeactivity of SAT an assay was used by following a method of Kredich andTomkins (1966). This method is based on a disulfide interchange betweenCoA, liberated from ace tyl-residue during the SAT catalyzed reaction,and 5,5′-dithio-bis-(2-nitro-benzoic acid). The formation of CoA wasassayed in 50 mM Tris-HCl (pH 7,6) containing 1 mM5,5′-dithio-bis-(2-nitro-benzoic acid), 1 mM EDTA, 20 mM L-serine and100 μM acetyl-CoA. The reaction was started by the addition of 10 μl ofcrude leaf extract (1,5 μg/μl total protein), the incubation temperaturewas 25° C. The production of thionitrobenzoic acid was monitored at 412nm in an spectrophotometer (Ultraspec 2000, Pharmacia Biotech, Uppsala,Sweden) against a blank control containing all materials exceptL-serine. A calibration curve was established with control solutionscontaining all materials and different concentrations of CoA (0-200nmol/ml). The activity assay was repeated independently with differentvolumes of crude leaf extract, i.e. 20, 40 and 60 μl. The analysis ofSAT activity in crude leaf extracts revealed that potato plantsexpressing the E. coli gene convert serine to OAS much more efficientlythan do the nbntransformed control plants (FIG. 1b), suggesting that thetransformed plants possess increased SAT activity, which is probably dueto the foreign E. coli serine acetyltransferase. Despite the strongincrease in SAT activity in the leaves of transgenic plants, no dramaticchange in the phenotype of these plants was visible with only oneexception: the transformant 48 showed a reduced apical dominanceresulting in a bushy phenotype. Interestingly the transformant 48 hadthe highest SAT activity from all transgenic plants.

EXAMPLE 2 Expression of the cysE Gene Leads to an Increase in theEndogenous Levels of Cysteine and Glutathione

Cysteine biosynthesis in plants takes place via a two step reaction. Theformation of cysteine from sulfide and O-acetyl-L-serine is catalyzed byO-acetylserine(thiol)lyase. O-acetyl-L-serine is synthesized by serineacetyltransferase from acetyl-coenzyme A and serine (Brunold andRennenberg, 1997). To investigate whether the expression of the cysEgene in potato plants influences the endogenous levels of cysteine, theconcentration of this sulfur containing amino acid in nontransformed andtransformed plants was determined. Thiols were prepared as described byRüegsegger and Brunold (1992). Seperation and quantification wereperformed by reverse-phase HPLC after derivatization withmonobrnmobimane according to Newton et al. (1981). As a modification,reduction of disulfides was done with bis-2-mercaptoethylsulfone and thelabelling reaction with monobromobimane was stopped with 15% HCl.

Frozen leaf material was homogenized to a fine powder and then extracted20 min in 0,1 N HCl (2 ml/0,2 gfw) at 4° C. After centrifugation of themixture at 4° C. (20 min, 14.000 g), 120 μl of the supernatent wereadded to 200 μl of 0,2 M 2-(cyclohexylamino)ethanesulfonic acid (pH9,3). Reduction of total disulfides was performed by adding 10 μlbis-2-mercaptoethylsulfone in 9 mM Tris-HCl, 5 mM EDTA (pH 8). After thereaction time of 40 min at room temperature, free thiolgroups werelabelled with monobromobimane. To this end, 20 μl of 15 mMmonobromobimane in acetonitrile were added to the mixture and kept for15 min in the dark at room temperature. The reaction was stopped byadding 250 μl 15% HCl. After keeping on ice for two hours in the dark,the reaction mixture was again centrifuged at 4° C. (10 min, 14.000 g).For cysteine and glutathione analysis, the supernatent was suitablydiluted with 0,1 N HCl. The samples were analysed according to themethod of Schupp and Rennenberg (1988) on a reverse phase HPLC column(C₁₈, 250×4 mm, 5 μm particle size, Macherey-Nagel, Oensingen,Switzerland). A solvent system consisting of 10% methanol; 0,25% aceticacid, pH 3,9 (NaOH) and 90% methanol; 0,25% acetic acid with a flow rateof 1,5 ml/min was used. Chromatography was followed by fluorescencedetection (excitation: 380 nm, emission: 480 nm, SFM 25 fluorescencedetector, Kontron, Zürich, Switzerland). Chromatograms were quantifiedby integration of peak areas. For cysteine analysis the twotransformants with the highest SAT-activity were used, i.e. SAT-48 andSAT-26 (see FIG. 2b). To this end, young and green leaves of 5 weeks oldplants were harvested and extracted, and the cysteine content wasdetermined via HPLC. Transgenic potato plants expressing the cysE genefrom E. coli exhibited significantly increased levels of cysteine (FIG.3a). The levels of the transformant SAT-48 were nearly threefold (45±10nmol per gram fresh weight of leaf tissue) and of the transformantSAT-26 twofold higher (33±6 nmol/gfw) than those amounts found innontransformed control plants (17±3 nmol/gfw), indicating that theexpression of cysE leads to an increase in the endogenous levels of theamino acid cysteine.

One of the major sinks of cysteine produced by thesulfate-assimilation/reduction cascade is the formation of glutathione,a tripeptide consisting of the aminoacids glutamate, cysteine andglycine. Because the transgenic potato plants expressing the E. coli SATcontained more cysteine, it is possible that this could have an effecton the biosynthesis of glutathione, keeping in mind that cysteine is onesubstrate for glutathione production. To investigate whether this is thecase, the levels of glutathione in leaves of the transgenic lines SAT-48and SAT-26 and of wild type plants were analysed. These measurementsrevealed that both transformants had significantly elevated glutathionelevels, being up to twofold higher (500-600 nmol/gfw) than in wild typeplants (300-350 nmol/gfw; FIG. 3b). This suggests that increased levelsof cysteine stimulate glutathione biosynthesis.

Taking into account that one molecule glutathione contains one moleculecysteine and that the total molar amounts of glutathione in potatoleaves are over 10-fold higher than the molar amounts of the free aminoacid cysteine, one can conclude that the synthetic capacity for cysteinein the transgenic potato plants is much stronger increased than only twoor threefold as could be thougth by only looking at the levels of freecysteine. An absolute increase of 200-300 nmol glutathione/gfw in thetransgenic plants is therefore equivalent with an approximately 10 to 18fold increased cysteine biosynthetic capacity, when having about 15-20nmol cysteine/gfw in leaves of wildtyp plants. Add to this the increasedlevels of free cysteine in the transgenic plants by about two orthreefold, the cysteine biosynthesis in the transformants is up to 20fold upregulated as compared to control plants.

EXAMPLE 3 Increased Endogenous Levels of Cysteine and Glutathione do notInfluence the Expression Pattern of OAS-TL Isoforms

A metabolically significant regulation of SAT activity by allostericinhibition of cysteine has been reported for the enzyme from watermelon(Saito, 1995). Bacterial SATs are on transcriptional level feed backinhibited by micromolar concentrations of cysteine. In contrast in asituation of cysteine limitation, the expression of bacterial SATs isstimulated (Kredich, 1987). Additionally, O-acetylserine(thiol)lyase,which is the enzyme directly following after the SAT in the cysteinebiosynthesis reaction cascade, is also regulated by cysteine ontranscriptional level. The expression of different cDNAs encoding forcompartment specific isoforms of O-acetylserine(thiol)lyase fromArabidopsis was observed to be stimulated in plants grown with limitedsulfate supply (Hell, 1994; Barroso, 1995; Hesse, 1997). Also in spinachthe expression of O-acetylserine(thiol)lyase isoforms is slightlyupregulated under sulfur-starved conditions (Takahashi and Saito, 1996).To investigate whether the enhanced levels of cysteine in the transgenicpotato plants expressing the cysE gene from E. coli influences theexpression pattern of the endogenous potato OAS-TL, leaf samples weretaken from transgenic and nontransformed plants and subjected to RNAblot analysis; see Example 1. For radioactive labelling of the potatoOAS-TL cDNAs (Hesse and Höfgen, 1998), DNA was cut with the appropriaterestriction enzymes and seperated on a 1% agarose gel. The DNA fragmentswere isolated from the gel using the “NucleoSpin Extract” kit fromMacherey-Nagel (Düren, Germany). This analysis revealed that althoughthe transgenic plants contained significant more cysteine in theirleaves, potato OAS-TL genes (both a cytosolic and a chloroplastidicisoform, Hesse and Högen, 1998) were not altered in their transcriptionrate compared to the expression in wild type plants (FIG. 3). Thissuggests that the increased levels of cysteine in the transgenic potatoplants have no detectable influence on the expression pattern of thepotato OAS-TL on transcriptional level.

EXAMPLE 4 Increased Endogenous Levels of Cysteine do not Influence theActivity of O-acetylserine(thiol)lyase

OAS-TL is regulated on activity level by the sulfur state within thecell. The activity of a cytosolic O-acetylserine(thiol)lyase fromArabidopsis thaliana for example is activated by sulfur limitation(Barroso, 1995; Hesse, 1997). Increasing of specific activity by sulfurdepletion have also been observed in cultured tobacco and C. reinhardtiicells and in maize leaves (Bergmann, 1980; Passera and Ghisi, 1982;León, 1988). In contrast high concentrations of sulfur seem to decreaseOAS-TL activity. The enzyme from Datura innoxia for example is inhibitedby higher sulfide concentrations (Kuske, 1994). In C. reinhardtii cellsOAS-TL activity is inhibited not only by sulfide, but also by OAS andcysteine (León and Vega, 1991). To find out, whether the increased SATactivity and the altered levels of cysteine and glutathione in thetransgenic potato plants have an effect on the activity of OAS-TL, anactivity-assay for this enzyme was performed with crude leaf extracts.O-acetylserine(thiol)-lyase activity was assayed by measuring theproduction of L-cysteine. Each assay was started by the addition of 5 μlcrude leaf extract (1 μg/μl total protein). Reactions were conducted in50 mM K₂HPO₄/KH₂PO₄ (pH 7,5) in the presence of 5 mM DTT, 10 mMO-acetylserine and 2 mM Na₂S (total volume 100 μl) and allowed toproceed for 20 min at 25° C. They were stopped by addition of 50 μl 20%trichloroacetic acid, and then analysed for L-cysteine production byusing the Gaitonde reagent (Gaitonde, 1967). The cysteine content wasmonitored at 560 nm in a spectrophotometer (Ultraspec 2000, PharmaciaBiotech, Uppsala, Sweden) against a blank control containing allmaterials except O-acetylserine. Experiments were repeated three times.However, these measurements revealed no difference in the OAS-TLactivities between the leaf extracts of wild type and transgenic plants(FIG. 4). So one can speculate, that the higher levels of cysteine andglutathione in the transgenic plants not only have no detectable effecton gene expression level but also have no effect on OAS-TL activity.

EXAMPLE 5 Expression for Transgenic Potato Plants Containing E. coli SATand Plant CγS mRNA

Two transgenic lines expressing E. coli SAT (e.g. SAT-48 and SAT-26)were selected for superinfection with a binary plasmid constructcontaining a CγS cDNA e.g. potato under the control of the 35S- and B33promoter, respectively. The binary vector is a pBIN19-derivativepermitting e.g. hygromycine resistance for plant selection. The plasmidswere introduced into the transgenic lines SAT48 and -26, respectivelyvia Agrobacterium tumefaciens as described by Rocha-Sosa, (1989).Selection conditions were chosen as described under Example 1.Superinfected transgenic lines expressing E. coli SAT and CγS werescreened on RNA level (Northern Blot), protein level (Western Blot) andenzymatic activity. Northern Blot experiments were performed asdescribed under Example 1. Lines with high CγS expression were selectedfor protein content and enzymatic activity. 10 μg protein of each leafextract were tested in Western Blot for increased protein content withrespect to wild type and original used transgenic line. Lines withincreased protein content were additionally tested for enzymaticactivity. 10 μg, 25 μg, 50 μg and 100 μg leaf extract were incubatedtogether with 10 μCi ³⁵S-Cysteine and 10 mM Succinylhomoserine for 30min at 30° C. in a total volume of 200 μl of 50 mM Tris/HCl, pH 7.8 and10 mM DTT. Reactions were stopped by addition of 50 μl 20% TCA. Afterneutralization and centrifugation 5 μl of each supernatant were analyzedby thin layer chromatography. A mixture of methanol/acetic acidethylester/H₂O=60:30:10) was used as running solvent. Transgenic plantswith high activity are further analyzed for GSH, CγS and Met content.

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2 1 30 DNA Artificial Sequence EcSAT-N Synthetic oligonucleotide used toamplify SAT from E. coli genomic DNA 1 gagagaccat ggcgtgtgaa gaactggaaa30 2 30 DNA Artificial Sequence EcSAT-C Synthetic oligonucleotide usedto amplify SAT from E. coli genomic DNA 2 gagagatcta gattagatcccatccccata 30

What is claimed is:
 1. A recombinant DNA molecule comprising (a) anucleic acid molecule encoding a protein having serineacetyl-transferase (SAT) activity, and optionally (b) a nucleic acidmolecule encoding a protein having cysteine-γ-synthase (CγS) activity;wherein said nucleic acid molecule(s) are operably linked to regulatoryelements allowing the expression of the nucleic acid molecule(s) inplant cells.
 2. The recombinant DNA molecule of claim 1, wherein saidprotein having SAT activity is a serine acetyltransferase fromprokaryotes or archaebacteria.
 3. The recombinant DNA molecule accordingto claim 1 or 2, wherein the protein having CγS-activity iscysteine-γ-synthase from potato, tobacco, tomato, rape seed orArabidopsis.
 4. The recombinant DNA molecule of claim 3, wherein thenucleic acid molecule of (a) and/or (b) is operably linked to anucleotide sequence encoding a transit peptide capable of directing theprotein(s) into a desired cellular compartment.
 5. The recombinant DNAmolecule of claim 4, wherein said cellular compartment is a plastid. 6.The recombinant DNA molecule of claim 1, wherein said regulatoryelements comprise a promoter active in plant cells.
 7. The recombinantDNA molecule of claim 6, wherein said promoter is inducible,constitutively expressed and/or is a cell, tissue or organ specificpromoter.
 8. The recombinant DNA molecule of claim 7, wherein saidpromoter is tuber-specific, seed-specific, endosperm-specific,embryo-specific, or phloem-specific.
 9. A vector comprising at least onerecombinant DNA molecule of claim
 1. 10. The vector of claim 9 furthercomprising a selectable marker.
 11. A transgenic plant cell comprisingstably integrated into its genome at least one recombinant DNA moleculeof claim 1 or at least one vector of claim 8 or
 9. 12. The transgenicplant cell of claim 11, comprising a nucleic acid molecule as defined inclaim 1(b) encoding a protein having cysteine-γ-synthase (CγS) activity.13. The transgenic plant cell of claim 11, comprising a selectablemarker.
 14. A transgenic plant or plant tissue comprising plant cells ofclaim
 11. 15. The transgenic plant of claim 14, wherein the level ofglutathione, cysteine and/or methionine is increased compared to a wildtype plant.
 16. Harvestable parts of a plant of claim 14 or
 15. 17.Propagation material of a transgenic plant of claim 14 or
 15. 18. Amethod for the production of transgenic plants which display anincreased level of glutathione, cysteine and/or methionine whichcomprises transforming a plant with at least one recombinant DNAmolecule of claim
 1. 19. The method of claim 18, wherein said increasedlevel of methionine or cysteine results in accelerated maturationprocesses, altered flowers and/or pathogen resistance.
 20. A method oftransforming a plant comprising: (a) introducing the recombinant DNAmolecule according to claim 1 or 2 into the genome of a plant, plantcell or plant tissue.